T cells respond to antigens in the context of major histocompatability complex (xe2x80x9cMHCxe2x80x9d) molecules. Cytotoxic T cells respond to antigens in the context of MHC class I molecules, while helper T cells respond to antigens in the context of MHC class II molecules (for review, see Davies, H., 1997, Introductory Immunobiology, Chapman and Hall, New York, pp. 177-223). Class I molecules are comprised of a heavy xcex1 chain and a xcex22-microglobulin light chain; class II molecules are heterodimers comprised of xcex1 and xcex2 chains, each having two domains and being of approximately the same length. The DNA regions containing MHC genes have been well characterized for mouse and man. The mouse MHC is referred to as the xe2x80x9cH-2 complexxe2x80x9d and the human MHC is referred to as the xe2x80x9cHLA complexxe2x80x9d (for Human Leukocyte Antigen). Class I molecules are encoded at the A, B and C loci in man and the K, D, and L loci in mouse. Class II molecules are encoded at the DP, DQ and DR regions in man and the I-A and I-E regions in mouse. At each region, a multitude of alleles has been identified.
The interaction between MHC-peptide complexes expressed on antigen presenting cells (APC) and T cell receptors (TCR) expressed on T cells leads to various T cell functions including proliferation and cytokine secretion, differentiation toward various cell subsets, anergy and apoptosis. See Davis et al. (1998) Ann. Rev. Immunol. 16:523-544. Various attempts have been made to mimic these immunomodulatory effects with soluble MHC-peptide complexes. For example, MHC molecules have been extracted from cell membranes and subjected to peptide elution followed by exchange for specific peptides in vitro. (Nag et al. (1996) Cell. Immunol. 170:25-33; Sharma et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88:11465-11469; Spack et al. (1995) J. Autoimmunity 8:787-807). Also, MHC molecules have been produced recombinantly and then loaded with peptides in vitro (Abastado et al. (1995) J. Exp. Med. 182: 439-447; Altman et al. (1996) Science 274: 94-96; Godeau et al. (1992) J. Biol. Chem. 267:24223-24229; Scheirle et al. (1992) J. Immunol. 149:1994-1999; Scott et al. (1996) J. Exp. Med. 183:2087-2095; Stem et al. (1992) Cell 68:465-477). Other attempts include the production of genetically engineered, covalently-linked peptide/MHC chimeras (International Patent Application Publication No. WO95/23814; Kozono et al. (1994) Nature 369:151-154; Mottez et al. (1995) J. Exp. Med. 181:493-502; Rhode et al. (1996) J. Immunol. 15:4885-4891; and U.S. Pat. No. 5,869,270).
A major disadvantage of monovalent MHC II-peptide ligands is that they are recognized by cognate TCRs with low avidity. The on-rates at 25xc2x0 C. vary from very slow (1,000) to moderately fast (200,000), whereas the off-rates are in a relative narrow range (0.5-0.01), or to a txc2xd of 12-30 seconds. Rates are estimated to be 2-3 times faster at physiologic temperature (Matsui et al. (1991) Science 254:1788-1791; Matsui et al. (1994) Proc. Nat. Acad. Sci. USA 91:12862-12866). In general, TCR exhibits 2 to 3-times lower binding affinity for the monovalent MHC II-peptide complex than for clonotypic antibodies to MHC-peptide complexes (Porgador et al. (1997) Immunity 6:715-726; Dadaglio et al. (1997) Immunity 6:727-738).
Recently, multivalent MHC II-peptide ligands with increased avidity for the cognate TCRs have been generated. A soluble bivalent MHC-II peptide ligand on an immunoglobulin scaffold, which binds stably and specifically to cognate TCR on T-cells, has been engineered. (Casares et al. (1997) Protein Engineering 10:1295-1301; International Patent Application Publication No. WO99/09064). Bivalent MHC II-peptide ligands engineered on an immunoglobulin scaffold exhibit approximately 20 to 25 times lower off-rates than the monovalent forms (Appel et al. (2000) J. Biol. Chem. 275:312-321). U.S. Pat. No. 6,211,342 discloses divalent MHC complexes that may be loaded with a peptide. Reich et al. expressed a BirA-dependent biotinylation site on xcex2-chain of MHC class II molecules to engineer tetravalent MHC II-peptide ligands through the streptavidin-mediated cross linking (Reich et al. (1997) Nature 387:617-620). Tetravalent MHC II/peptide ligands were successfully used to identify low-frequency antigen-specific T-cells in the peripheral blood of patients with HIV infection (Crawford et al. (1998) Immunity 8:675-682). However, the tetravalent MHC II/peptide ligands did not exceed the avidity of immunoglobulin-based, dimeric MHC II/peptide ligands, presumably because of the rigidity of biotin-streptavidin bonds that may not provide optimal accommodation of the tetramers on the TCR motifs.
Although the tetrameric MHC II-peptide molecules generated through the biotin-streptavidin bonds are valuable tools for in vitro investigation, the non-covalent nature of this bonds raises the concern of its stability in vivo. Accordingly, there is a need in the art for multimeric MHC II-peptide molecules that maintain structural integrity in vivo and exhibit immunomodulatory effects on T cells.
The present invention provides a complex comprising at least two chimeric molecules, wherein each chimeric molecule comprises an immunoglobulin constant region element having two heavy chains, wherein each heavy chain is linked to an MHC element, and wherein a peptide of interest is associated with each of the MHC elements, and wherein at least two of the chimeric molecules are covalently linked through a carbohydrate residue of the immunoglobulin constant region element by a polyalkylene glycol linker.
The present invention further provides compositions comprising the complexes.
In another embodiment, the present invention is directed to a method of making such a complex.
The present invention also provides a method of modulating T cell function comprising administering the complex of the invention, and a method of diagnosing an autoimmune disorder using the complex of the invention.